Twisted states of Bacillus subtilis macrofibers reflect structural states of the cell wall.

Abstract

Static and dynamic studies of helical Bacillus subtilis macrofibers reveal that a spectrum of twisted states exists ranging from tight left-handed structures with twist equal to ='40 left turns per mm to tight right-handed structures with twist equal to 57 right turns per mm. In the lyticdeficient strain FJ7, twist varies as a function of growth temperature above or below 39°C, where there is zero twist. The relationship between the temperature (below 390C) at which right-hand structures are produced to the time it takes for them to begin the inversion process in which they become lefthanded following transfer to 480C reveals that structures with less twist are more rapidly converted to left-handedness than are those with higher values of twist. The initial response of live macrofibers to digestion by lysozyme consists of "relaxation" motions in which the twist of both left-and right-handed structures changes towards the right-hand end of the spectrum. The rate of relaxation is =-5-fold higher at the left-hand end than at the right-hand end. These findings suggest that cell wall polymers can assume a range of structural states during helical growth and that these determine the quantitative aspects of macrofiber shape as well as the sensitivity of walls to attack by lysozyme.The shape deformation responsible for the production of helical macrofibers of Bacillus subtilis has been interpreted as a ramification of the organization of the cell wall and the interplay of forces in the cell wall associated with growth (1, 2). Key features of macrofiber behavior are failure of cell separation leading to formation of cellular filaments, twisting about the cylindrical axis of the cells during growth, whiplike motions of filaments and subsequently fibers, which eventually cause a given fiber to touch itself, and folding of the structure by the twisting together of the two arms so created. The folding process is repeated numerous times resulting in the construction of a macrofiber in which all of the cellular filaments are packed into an organized structure of the same helix hand (3).Macrofibers can exist as either right-or left-handed structures (2). In this communication we report that a spectrum of macrofiber states exists in which the twist ranges from rightthrough neutral to left-handed. For any given strain, the position in the spectrum depends on the growth medium, the concentration of divalent cations, and temperature, but for any set of conditions the structures grow with constant twist. All cells within a given structure must therefore assemble their walls following the same rules of geometry.In some strains macrofibers can be made to invert their helix hand (4). We report here the kinetics of temperatureinduced inversion in one of themn. The twist of macrofibers can also be changed as the result of attack by enzymes. The cell wall of B. subtilis consists primarily of two polymers: peptidoglycan and teichoic acid (5). In this paper we show that cleavage by lysozyme of the peptidoglycan backbone in live macr...

Regulation of Bacillus subtilis macrofiber twist development by ions: effects of magnesium and ammonium

“…Both systems reveal that electrostatic factors affect cell wall assembly, which in turn affects cell morphology. In the macrofiber system, however, a biomechanical factor has been shown to also be important (14,16). The preferential response of macrofibers to ammonium rather than magnesium in strains such as FJ7 (Table 4) is also without precedent, although it has been reported that ammonium limitation of B. subtilis leads to differences in cell wall polymer composition, resulting in cells that are highly autolytic and extremely sensitive to lysozyme (3).…”

“…We suggested that macrofiber twist states reflect structural states of the cell wall (16) (2,3,8,10,13). In the macrofiber system we found by using a temperature-pulse system that a left factor protein(s) associated with the cell surface is required for the establishment of left-handed twist states (4).…”

“…Cultivation of macrofibers has been described previously (15). Macrofiber twist, helix hand inversion, and lysozyme-induced relaxation motions have been defined previously (5,6,15,16,18).…”

Cell wall mechanical properties as measured with bacterial thread made from Bacillus subtilis

“…B. subtilis 168 FJ7, a cell separation-suppressed (lyt) mutant that carries lyt-2 and metC markers, was used to produce the cultures from which bacterial threads were made. It is the same strain studied previously as bacterial thread (22) and also as macrofibers (15,16).…”

“…B. subtilis 168 FJ7, a cell separation-suppressed (lyt) mutant that carries lyt-2 and metC markers, was used to produce the cultures from which bacterial threads were made. It is the same strain studied previously as bacterial thread (22) and also as macrofibers (15,16).Media and growth conditions. Cultures were grown in the complex medium TB consisting of the following (in grams per liter of water): tryptose (Difco Laboratories), 10; beef extract (Difco), 3; and NaCl, 5.…”